Syk and Fyn are required by mouse megakaryocytes for the rise in intracellular calcium induced by a collagen-related peptide

Stimulation of platelets by collagen leads to activation of a tyrosine kinase cascade resulting in secretion and aggregation. We have recently shown that this pathway involves rapid tyrosine phosphorylation of an Fc receptor gamma chain, which contains an immunoreceptor tyrosine-based activation motif (ITAM), enabling interaction with the tandem SH2 domains of the tyrosine kinase Syk. Activation of Syk lies upstream of tyrosine phosphorylation of phospholipase Cgamma2. In the present study we sought to test directly the role of the ITAM/Syk interaction and the role of the Src-related kinases in collagen receptor signaling using mouse megakaryocytes. We demonstrate that the calcium-mobilizing action of a collagen-related peptide (CRP) is kinase-dependent, inhibited by the microinjection of the tandem SH2 domains of Syk and abolished in Syk-deficient mice. Furthermore, the CRP response is abolished by the Src family kinase inhibitor PP1 and inhibited in Fyn-deficient mice. In contrast, the calcium response to the G-protein-linked receptor agonist thrombin is not significantly altered under these conditions. These results provide direct evidence of the functional importance of Fyn and Syk in collagen receptor signaling and support the megakaryocyte as a model for the study of proteins involved in this pathway.     

Effect of 5-hydroxytryptamine on intracellular calcium dynamics in cultured rat vascular smooth muscle cells

The present study elucidated the precise mechanism of 5-hydroxytryptamine (5-HT)-induced increase of intracellular Ca2+ concentration ([Ca2+]i) in cultured vascular smooth muscle cells isolated from rat aortic media. [Ca2+]i was measured using fluorescent Ca2+ indicator, fura-2. 5-HT caused a dose-dependent increase in [Ca2+]i, which was completely inhibited by ketanserin. alpha-Methyl-5-HT had an equipotent effect to 5-HT. Diltiazem at 10 microM partially suppressed the 5-HT-induced increase in [Ca2+]i. 5-HT also augmented Mn2+ influx, when monitored by Mn2+ quenching of fura-2 fluorescence. When extracellular Ca2+ (1.3 mM) was removed, a decrease in resting level and a small, transient increase in [Ca2+]i were observed. 5-HT stimulation also induced an increase in the production of inositol triphosphate. 5-HT-induced increase in [Ca2+]i was significantly, but partially inhibited by staurosporin and H-7. Phorbol 12-myristate 13-acetate induced an increase in [Ca2+]i, which was abolished by removal of extracellular Ca2+. 5-HT-induced increase in [Ca2+]i was not affected by the pretreatment with pertussis toxin (PTX), and was not accompanied by a change in cyclic AMP content. These results suggest that, in cultured rat aortic smooth muscle cells, 5-HT increases [Ca2+]i via 5-HT2 receptor subtype by inducing influx of extracellular Ca2+ partially through L-type voltage-dependent Ca2+ channel, as well as by mobilizing Ca2+ from its intracellular stores. Activation of protein kinase C may be positively involved in the regulatory mechanism of Ca2+ influx, but PTX-sensitive G protein and cyclic AMP seem to be not involved.     

The response of mouse oocytes injected with sea urchin spermatozoa

Entrainment to the 24-hour light-dark cycle is of adaptive significance to mammals. Human infants are no exception, but some postnatal care habits prevalent in developed countries can interfere with the physiological mechanisms underlying circadian synchronization. We describe the physiological mechanisms of entrainment to the light-dark cycle in fetuses and newborns, and some common parental care behaviors which subject the developing circadian system of the newborn to conflicting temporal cues. Improvements in parental care are proposed which may improve the circadian synchronization of newborns, and their parents or caregivers.     

Infection of the laboratory mouse with the intracellular pathogen Ehrlichia chaffeensis

To determine the basis of susceptibility and resistance to human monocytic ehrlichiosis (HME), immunocompetent and immunocompromised mice were infected with Ehrlichia chaffeensis and bacterial loads were measured by PCR and by immunohistochemistry. Immunocompetent (C. B-17 and C57BL/6) mice cleared the bacteria within 10 days, but immunocompromised SCID and SCID/BEIGE mice developed persistent infection in the spleen, liver, peritoneal cavity, brain, lung, and bone marrow and became moribund within 24 days. Both immunocompromised strains lack T and B lymphocytes, but the SCID/BEIGE strain is also deficient in natural killer (NK) cell function. During advanced stages of disease, the infections were associated with wasting, splenomegaly, lymphadenopathy, liver granulomas and necroses, intravascular coagulation, and granulomatous inflammation. Histochemical and immunohistochemical localization studies confirmed the presence of bacteria in tissues, and viable bacteria were cultured from infected animals. The data reveal that T and/or B cells play an essential role during resistance of immunocompetent mice to infection with E. chaffeensis and demonstrate the utility of immunocompromised mice as an experimental model for the study of HME.     

Upregulation of CD44 in the inflamed mouse air pouch injected with synthetic lipid A

OBJECTIVE: To investigate aspects of the inflammatory process of the mouse subcutaneous air pouch -- a facsimile synovial cavity -- induced by injection of lipid A, and to determine the expression and upregulation of CD44 in the lining cell layer of the inflamed air pouch. METHODS: Histological changes of inner walls in the mouse air pouch were evaluated 1, 3, and 7 days after injection of lipid A. RESULTS: Polymorphonuclear cell infiltration in the lining layer reached the maximum one day after injection of 10 microg of lipid A (10/10 mice in Grade 3; p < 0.01), while mononuclear cell infiltration and lining cell hyperplasia reached the maximum 3 days after injection (5/10 mice in Grade 2; 5/10 in Grade 3; p < 0.05; 39+/-11 layers, p < 0.05, respectively). The number of cell depth of CD44 positive lining layers and interleukin 1alpha (IL-1alpha) positive lining layers reached the maximum 3 days after injection (39+/-7 layers, p < 0.01; 35+/-12 layers, p < 0.05, respectively). CONCLUSION: These findings suggest that CD44 may have some connection with the proinflammatory cytokine IL-1alpha and induce inflammatory responses in the air pouch injected with lipid A.     

Relationship between intracellular free calcium concentrations and the intracellular development of Toxoplasma gondii

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.     

ATP in iron overload-induced intracellular calcium changes

The molecular signaling events by which leptin exerts its functions in vivo are not well delineated. Here, we show a novel leptin signaling mechanism that requires phosphoinositide 3-kinase (PI 3-kinase)-dependent activation of cyclic nucleotide phosphodiesterase 3B (PDE3B) and subsequent suppression of cAMP levels. In pancreatic beta cells, leptin causes the activation of PDE3B, which leads to marked inhibition of glucagon-like peptide-1-stimulated insulin secretion. The effect of leptin is abolished when insulin secretion is induced with cAMP analogues that cannot be hydrolyzed by PDE3B. Selective inhibitors of PDE3B and PI 3-kinase completely prevent the leptin effect on insulin secretion and cAMP accumulation. The results demonstrate that one of the physiological effects of leptin, suppression of insulin secretion, is mediated through activation of PDE3B and suggest PDE3B as a mediator of leptin action in other tissues.     

Spatiotemporal dynamics of clotting and pattern formation in human blood

We examined the spatial dynamics of in vitro clot growth in human blood and plasma and found that initially, a clot grows at a constant speed, then abruptly stops and becomes surrounded by an 'inhibition zone' in which coagulation is strongly suppressed. We also observed the formation of 'stratified structures' (target patterns) in which solid layers alternated with liquid plasma. These and other spatial regimes of clotting are explained in terms of two interacting concentration waves propagating without attenuation. The experimental results are consistent with a hypothesis that blood is a bi-excitable medium, a new type of excitable medium.     

Inhibition of phosphoinositide metabolism or chelation of intracellular calcium blocks FSH-induced but not spontaneous meiotic resumption in mouse oocytes

Mammalian oocytes are arrested at the diplotene phase of the first meiotic division until ovulation. In the mouse, germinal vesicle breakdown (GVBD) and progression to metaphase II is thought to be triggered by a positive signal originating in the follicular cells following stimulation by the luteinizing hormone (LH) surge. Isolated, fully grown oocytes can also undergo spontaneous reinitiation of meiosis in vitro in the absence of gonadotrophin stimulation. To investigate the mechanism of meiotic resumption, inhibitors of phosphoinositide metabolism and an intracellular calcium chelator were used during maturation in vitro under different conditions. In a series of experiments, isolated cumulus cell-oocyte complexes (COCs) maintained in meiotic arrest by hypoxanthine were induced to resume meiosis by treatment with follicle-stimulating hormone (FSH). Under these conditions, both LiCl and neomycin, which inhibit phosphoinositide hydrolysis, produced a dose-dependent inhibitory effect on meiotic resumption. Similar results were obtained when FSH-induced meiotic resumption was observed in the presence of the acetoxymethyl ester form of 1, 2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA/AM), an intracellular calcium chelator. In hypoxanthine-arrested oocytes, GVBD induced by epidermal growth factor (EGF), which mimics FSH action in in vitro maturation, was also repressed by LiCl and neomycin. Conversely, meiotic resumption triggered by a pulse of 8-bromo-cyclic adenosine monophosphate (8-Br cAMP) was not affected by these two inhibitors. In experiments in which oocytes were cultured under conditions which permit spontaneous meiotic maturation, resumption of meiosis was not affected by either inhibition of phosphoinositide hydrolysis or chelation of intracellular calcium. Therefore, it appears that meiotic resumption induced by hormone stimulation requires activation of the phosphoinositide pathway and mobilization of intracellular calcium. In contrast, spontaneous maturation probably occurs through a different mechanism because it is not affected by inhibition of this signaling pathway.     

Involvement of intracellular calcium in morphine tolerance in mice

We attempted to mimic in small upflow anaerobic sludge bed (UASB) bioreactors the metabolic association found in nature between methanogens and methanotrophs. UASB bioreactors were inoculated with pure cultures of methanotrophs, and the bioreactors were operated by using continuous low-level oxygenation in order to favor growth and/or survival of methanotrophs. Unlike the reactors in other similar studies, the hybrid anaerobic-aerobic bioreactors which we used were operated synchronously, not sequentially. Here, emphasis was placed on monitoring various methanotrophic populations by using classical methods and also a PCR amplification assay based on the mmoX gene fragment of the soluble methane monooxygenase (sMMO). The following results were obtained: (i) under the conditions used, Methylosinus sporium appeared to survive better than Methylosinus trichosporium; (ii) the PCR method which we used could detect as few as about 2,000 sMMO gene-containing methanotrophs per g (wet weight) of granular sludge; (iii) inoculation of the bioreactors with pure cultures of methanotrophs contributed greatly to increases in the sMMO-containing population (although the sMMO-containing population decreased gradually with time, at the end of an experiment it was always at least 2 logs larger than the initial population before inoculation); (iv) in general, there was a good correlation between populations with the sMMO gene and populations that exhibited sMMO activity; and (v) inoculation with sMMO-positive cultures helped increase significantly the proportion of sMMO-positive methanotrophs in reactors, even after several weeks of operation under various regimes. At some point, anaerobic-aerobic bioreactors like those described here might be used for biodegradation of various chlorinated pollutants.     

New substituted 1,4-benzoxazine derivatives with potential intracellular calcium activity

Substituted 1,4-benzoxazines bearing an amino side chain at the 2-position were prepared and were found to have a moderate activity on intracellular calcium. Of the compounds studied it was found that those which possess a homoveratrylamino moiety exhibited superior potency. The chain length and the nature of the amine (4-fluorophenylpiperazine, 4-fluorobenzhydryloxyethylamine, N-substituted homoveratrylamine) is discussed. The 4-benzyl-3, 4-dihydro-2-[3-[[2-(3,4-dimethoxyphenyl)ethyl]amino]propyl]-2H-1, 4-benzoxazine (3c) is the most potent derivative of the series with a ratio of IC50 values against PE (phenylephrine) and K+ of 2.1. Under these test conditions a ratio near 1 indicates potential intracellular calcium activity while a ratio greater than 100 an action on extracellular calcium influx.     

Relationship between intracellular calcium store depletion and calcium release-activated calcium current in a mast cell line (RBL-1)

The kinetic relationship between depletion of endoplasmic reticulum calcium stores and the activation of a calcium release-activated calcium current (Icrac) was investigated in the RBL-1 mast cell line. The inositol trisphosphate receptor activator, inositol 2,4, 5-trisphosphate ((2,4,5)IP3), the sarcoplasmic-endoplasmic reticulum calcium ATPase inhibitor, thapsigargin, and the calcium ionophore, ionomycin, were used to deplete stored calcium. For (2,4,5)IP3 and thapsigargin, a significant delay was observed between the initiation of calcium store depletion and the activation of Icrac. However, for ionomycin, little or no delay was observed. This may indicate that a specialized subcompartment of the endoplasmic reticulum functions as a regulator of calcium entry and that this compartment is relatively resistant to depletion by (2,4,5)IP3 and thapsigargin but not to depletion by ionomycin. For all three calcium-depleting agents, the rate of development of Icrac, once initiated, was relatively constant, suggesting an all-or-none mechanism. However, there were also clear experimental situations in which submaximal, graded depletion of stored calcium resulted in submaximal activation of Icrac. This complex behavior could also result from the existence of a specific subcompartment of endoplasmic reticulum regulating Icrac. The kinetic behavior of this compartment may not be accurately reflected by the kinetics of calcium changes in the bulk of endoplasmic reticulum. These findings add to the growing body of evidence suggesting specialization of the endoplasmic reticulum calcium stores with regard to the control of capacitative calcium entry.